The primary goal of this project is to identify chemical probes that either increase or decrease the lipid content of cells. Most, and probably all cells are capable of storing energy rich lipids (mainly triacylglycerols), which are generated on basis of de novo synthesized fatty acids or non-esterified free fatty acids (NEFA) taken up from the environment. Storage lipids are deposited in specialized organelles, the so-called lipid droplets (LDs). LDs are thought to arise at the endoplasmic reticulum in a budding-like process. Once released into the cytoplasm, LD volume increases by targeted lipogenesis or fusion of existing droplets, suggesting extensive trafficking events. Remobilization of LD stores involves enzymatic breakdown of the storage lipids by lipases. Lipogenesis and lipolysis needs to be delicately fine-tuned, as diminished or increased lipid stores have dramatic consequences for the organism, as demonstrated in human diseases including lipodystrophy, atherosclerosis or obesity. There are only few drugs for treating metabolic diseases and a very limited number of chemical probes to study lipid storage in vitro. We found that also embryonic Drosophila S3 and Kc167 cells are capable of depositing LDs and developed an assay to measure lipid storage amounts by fluorescent staining of LDs and cells and microscopic or laser-scanning microplate cytometry based detection. In a genome-wide RNA interference (RNAi) screen, the assay identified about 500 gene-functions regulating lipid stores. The same assay was further optimized for small compound screening in S3 cells. Assay profiling confirmed its precision, robust response to known small-molecule modulators of lipid storage and suitability for miniaturization and HTS. Screening to date of 8,874 compounds has yielded a small number of actives, some of which are connected with protein targets previously associated with organismic lipid storage regulation. Further screening will increase the chance that potent compounds can be found with the potential of mechanistic insights and therapeutic development. Validation of candidate chemical probes during assay development in mouse AML12 hepatocytes demonstrated evolutionary conservation of our findings. Orthologous testing with RNAi-mediated knockdown experiments additionally provided epistasis information suitable to further characterize identified compounds. PUBLIC HEALTH RELEVANCE: The chemical probes yielded by the project should be useful tools in providing a better understanding of cellular and organismic lipid storage on a functional and evolutionary level. Furthermore, active substances might result in therapies treating emerging lipid storage associated diseases including atherosclerosis, diabetes or obesity. As well, the present assay will establish a profile of compounds within the MLSMR that modulate this ubiquitous area of biology.